Question

The following DNA sequence contains a bacterial gene:

atgcgccgta ggacacttga ttacgaacaa tttctacaaa acacttgata ctgtatgagg atacagtata attgcttcaa cagaacatat tgactatccg gcatgacagg agtaaaaatg atggctatcg acgaaaacaa acagaaagcg ttggcggcag cactgggcca gattgagaaa caatttggta aaggctccat catgcgcctg ggtgaagacc gttccatgga tgtggaaacc atctctaccg gttcgctttc actggatatc gcgcttgggg caggtggtct gccgatgggc cgtatcgtcg aaatctacgg accggaatct tccggtaaaa ccacgctgac gctgcaggtg atcgccgcag cgcagcgtga aggtaaaacc tgtgcgttta tcgatgctga acacgcgctg gacccaatct acgcacgtaa actgggcgtc gatatcgaca acctgctgtg ctcccagccg gacaccggcg agcaggcact ggaaatctgt gacgccctgg cgcgttctgg cgcagtagac gttatcgtcg ttgactccgt ggcggcactg acgccgaaag cggaaatcga aggcgaaatc ggcgactctc acatgggcct tgcggcacgt atgatgagcc aggcgatgcg taagctggcg ggtaacctga agcagtccaa cacgctgctg atcttcatca accagatccg tatgaaaatit ggtgtgatgt tcggtaaccc ggaaaccact accggtggta acgcgctgaa attctacgcc tctgttcgtc tcgacatccg tcgtatcggc gcggtgaaag agggcgaaaa cgtggtgggt agcgaaaccc gcgtgaaagt ggtgaagaac aaaatcgctg cgccgtttaa acaggctgaa ttccagatcc tctacggcga aggtatcaac ttctacggcg aactggttga cctgggcgta aaagagaagc tgatcgagaa agcaggcgcg tggtacagct acaaaggtga gaagatcggt cagggtaaag cgaatgcgac tgcctggctg aaagataacc cggaaaccgc gaaagagatC gagaagaaag tacgtgagtt gctgctgagc aacccgaact caacgccgga tttctctgta gatgatagcg aaggcgtagc agaaactaac gaagattttt aatcgtcttg tttgatacac aagggtcgca tgtgcggccc ttttgttttt ttaagttgta aggatatgcc atgacacgat

1. Copy the above sequence and indicate on it the:

a. -10 and -35 promoter region

b. Ribosome binding site

c. Stop codon (and whether it is an amber, opal or ochre codon),

d. Transcriptional terminator.

e. Note the atg start site has been marked and the gene is indicated in italics

2. Is the promoter a strong or weak promoter? Why?

3. Is the terminator a Rho dependent or Rho independent terminator? Why?

Answer 1

Consensus -10 and -35 sequences:

• The Promoter region of prokaryotes comprises of consensus sequences -10 and -35 upstream of transcription start site. The -10 sequence is A T rich and usually contain TATAAT. The -35 sequence contains TTGACA.
• The E. coli RNA polymerase (σ subunit), recognizes and binds to the -35 site. Followed by enzyme interaction and DNA unwinding at the -10 region.
• Eukaryotic promotor is tripartite, consisting of- GC box, CAT, TATA box. Initiation requires binding of transcriptional factors, like TFIIA, TFIIB, etc, binding to TATA box.

Ribosome binding site (RBS):

• Ribosome binding site (RBS) is nucleotide sequence present upstream (2 or 3 to 7 or10 nucleotides upstream) of start codon (AUG) in mRNA. This sequence is important (though not absolute essential) for initiation of translation in prokaryotes (like bacteria).

  This sequence, also referred to as Shine-Dalgarno’s (SD) sequence, comprises of 5^’ untranslated region (UTR) in mRNA and is purine rich. A complementary pyrimidine rich 3^’ sequence (anti-SD sequence) is present in the 16S rRNA of ribosomal subunit. This interaction between the SD sequence of rRNA and anti-SD sequence of mRNA plays an important role (in most cases) for initiation of translation in prokaryotes.

Terminator:

• Transcription is terminated at the terminator sequence.
• Terminator sequence signals the dettachment of the RNA polymerase.

Stop codon:

• Translation starts from the start codon sequence (AUG) and ends at Stop codons (UAG, UGA, UAA)
• UAG – Amber
• UAA- Ochre
• UGA- Opal

2.

a)

• The promoter is considered strong if they match the ideal consensus sequences, causing maximum interaction with RNA polymerase.or sigma factor.

• If the promoter has lesser match with the ideal sequence, the promoter is considered weak.

• Thus, the promoter is weak, as -10 and -35 sequences does not match the ideal consensus sequence.

b)

• Termination may be Rho dependent or Rho independent.
• In Rho dependent termination: Rho protein binds to the terminator sequence without secondary structure, hair- pin structures further formed to halt RNA polymerase.
• In Rho independent termination: the terminator sequence have series of adenine (A) or poly U (or) on RNA, along with inverted repeats, forming hair-pin structures.
• Thus the presence of inverted repeats with T indicates Rho independent termination.